Chimeric antigen receptor (CAR) T-cells graft the specificity of a monoclonal antibody (mAb) to a T-cell (Pule, M., Finney, H. & Lawson, A. Artificial T-cell receptors. Cytotherapy 5, 211-226 (2003)). CAR T-cells are usually autologous—i.e. they are generated from the patient's own lymphocytes. This is effective and simple but has a number of limitations: (1) it may be difficult or impossible to generate a product from patient's own lymphocytes due to insufficient quantity or quality of lymphocytes consequent to disease or chemotherapy; (2) there may be insufficient time to generate an autologous CAR T-cell product due to the tempo of the patient's illness; and (3) autologous production requires a bespoke product to be manufactured for each patient which makes manufacture costly.
An alternative approach is to generate “off-the-shelf” CAR T-cells from healthy donor lymphocytes. Cord blood lymphocytes are a particularly convenient source of donor lymphocytes for off-the-shelf CAR T-cell production. Using the off-the-shelf approach, production of the CAR T-cell product is independent of the patient. Furthermore, if the manufacturing process lends itself to economies of scale, the off-the-shelf approach may advantageously reduce the cost of production of the CAR T-cell product.
Given the wide variability of human leukocyte antigen (HLA) types, it is very likely that any off-the-shelf CAR T-cell product will be completely HLA-mismatched from the recipient. It is simply not feasible to have a HLA-matched, off-the-shelf CAR T-cell product ready for every recipient in need thereof. This HLA mismatch is associated with its own technical challenges, in particular graft versus-host disease (GVHD). In GVHD, that native T-cell receptor (TCR) of T-cells in donated tissue (the “graft”) recognise antigens in the recipient (the “host”) as foreign. Thus, transplanted T-cells attack host cells and tissues, causing damage to the host organs. Acute or fulminant GVHD, which normally occurs within the first 100 days following transplant, is associated with significant morbidity and mortality. Chronic GVHD, which normally occurs after 100 days following transplant, adversely influences long-term survival.
GVHD typically occurs in the setting of allogeneic haematopoietic stem cell transplantation (HSCT), in which the donor and recipient are fully or partially HLA-matched. In the off-the-shelf CAR T-cell approach, the CAR T-cell product and the recipient are completely HLA-mismatched. When the donor and recipient are not matched, more severe type of GVHD known as “transfusion-associated GVHD” (TA-GVHD) occurs.
In order to be of widespread utility, an off-the-shelf CAR T-cell product must cause, at most, a minimal amount of GVHD when administered to a HLA-mismatched recipient. Current approaches to attenuating the ability of HLA-mismatched CAR T-cells to cause GVHD involve editing the genome of the CAR T-cells to disrupt native TCR expression, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. These genome editing methods can disrupt a gene, entirely knocking out all of its output. However, several problems are associated with these genome editing approaches. Firstly, the genes required for these approaches typically have to be delivered separately to CAR T-cells during their production, for instance by electroporation with synthetic mRNA. Consequently, the resultant disruption or knockdown of the native TCR in the T-cell is not linked to CAR expression. This means that sorting for CAR-expressing T-cells does not necessarily also sort for T-cells expressing the genome editing genes required to disrupt native TCR expression. Likewise, sorting for expression of genome editing genes does not necessarily sort for CAR-expression. Therefore, to obtain CAR T-cells suitable for use in an off-the-shelf product, it is necessary to perform two different sorting steps, one to select for CAR-expressing T-cells and one to select for T-cells expressing the genome editing genes. Secondly, ZFNs, TALENS and CRISPR/Cas can all introduce off-target gene disruptions and cause unwanted translocations.
An improved method of disrupting the expression of the native TCR in CAR T-cells is therefore required.